Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens

• Published in: Journal of virological methods Vol. 57; no. 2; pp. 195 - 201
• Main Authors: HALE, A. D, GREEN, J, BROWN, D. W. G
• Format: Journal Article
• Language: English
• Published: London Elsevier 05.04.1996; Amsterdam; New York, NY
• Subjects: Base Sequence; Biological and medical sciences; Cetrimonium; Cetrimonium Compounds; Chelating Agents; Chromatography, Gel; Detergents; Dextrans; DNA, Viral; Feces - virology; Fundamental and applied biological sciences. Psychology; Guanidines; Humans; Microbiology; Molecular Sequence Data; Norwalk virus - genetics; Norwalk virus - isolation & purification; Polymerase Chain Reaction - methods; Resins, Synthetic; RNA, Viral - analysis; Sensitivity and Specificity; Techniques used in virology; Thiocyanates; Virology; Virus; Polymerase chain reaction; Purification; RNA; Caliciviridae; Reverse transcription; Method; Detection
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/0166-0934(95)01966-9

Four methods for extraction of the RNA genome of small round structured viruses (SRSVs) from faecal specimens by reverse transcription polymerase chain reaction (RT-PCR) were evaluated. The efficiency of recovery of viral RNA and removal of amplification inhibitors were compared. RNA extraction using the metal chelating agent Chelex-100 and Sephadex G200 column chromatography were the most sensitive, detecting a 10(-4) dilution of a known SRSV positive specimen. Guanidinium thiocyanate (GTC) with adsorption of viral RNA onto silica was 10-fold less sensitive. The fourth method, based on PEG precipitation followed by phenol/chloroform extraction with the addition of the detergent cetyltrimethylammonium bromide (CTAB), only detected a 1 in 10 dilution of the positive specimen. The CTAB method was 2- to 50-fold less sensitive than the GTC/silica method when dilution series of three further SRSV positive specimens were tested. Thirty-six SRSV negative faecal specimens were spiked with virus and RT-PCR performed following RNA extraction by each of the four techniques in order to assess the ability of these methods to remove amplification inhibitors. The GTC/silica method successfully removed inhibitors from all samples whereas partial or complete inhibition was observed in seven (19%) specimens following extraction by the CTAB method, 17 (47%) by the Sephadex method, and 20 (56%) by the Chelex method. We conclude that, of these four methods, the GTC/silica method is the most appropriate for the extraction of viral RNA from faecal samples prior to RT-PCR for detecting SRSVs.